Effects of rock wool on the lungs evaluated by magnetometry and biopersistence test

As an experimental material, we used an RW sample manufactured by NC Co., Ltd., Japan that was provided by the Rock Wool Association, Japan. Fluorescence X-ray spectroscopy showed that the RW used in the present study was chemically composed of SiO₂ 39%, CaO 33%, Al₂O₃ 14%, MgO 5.0%, Fe₂O₃ 1.8%, and S 0.6%.

Originally, RW is present in the form of lumps of different fiber sizes (both length and width). We adjusted the RW fiber size in accordance with the method of Kohyama et al. (1997) to obtain fiber samples of appropriate size for animal experiments [13]. RW fibers thus obtained were dispersed in an exposure chamber and the fiber sizes were measured. Their geometric mean length (geometric standard deviation, GSD) and geometric mean width (GSD) were 15.49 (2.02) μm and 2.44 (1.59) μm, respectively. Then, to make it easier to generate RW in the nose-only inhalation exposure system, the pressurized and pulverized RW fibers were mixed with glass beads (BZ-02, AS ONE Corporation, Osaka, Japan) at a weight ratio of 1 (RW) to 39 (glass beads).

Male Fischer 344 (F344) rats (6 to 10 weeks old; which is specifically recommended by EC Protocol, 1999) were used for each experiment. To acclimatize the rats to the environment of the laboratory, they were first housed in cages for about one week with free access to water and food. The temperature was kept at 22°C and 40% humidity, with a continuous supply of fresh filtered air.

In the lung magnetometric evaluation, an exposure group and a control group comprised 6 rats each. The study material (RW fibers) was supplied with air into the exposure chamber and exposed to the noses of rats of the exposure group in the same way as reported previously [14‐16]. The rats in the control group were not exposed to RW but underwent lung magnetometry only.

In the biopersistence test, 12 rats were used per experiment and the experimental was repeated twice, and in phathological evaluation, 12 rats were used (36 rats in total). The rats were exposed to RW fibers continuously for 6 hours daily for 5 consecutive days. Each day during the experimental period, the rats fixed in the upper rat holders of the main chamber were replaced by the rats in the lower rat holders, rotating the positions among the upper and lower rat holders.

Figure 1 shows an outlined view of the lung magnetometric evaluation apparatus. Magnetometric evaluation of lungs was performed in 6 rats each of RW-exposed and control groups according to the method reported by Aizawa et al. (1991). One day after exposure, rats were anesthetized by inhalation of diethyl ether. In this study triiron tetraoxide (Toda Kogyo Corp., Tokyo, Japan) was used as magnetic particles with the geometric mean particle size of 0.26 μm.

RW-exposed and control rats were intratracheally catheterized and instillated with 3 mg of triiron tetraoxide suspended in 0.2 mL of physiological saline one day after RW exposure. Each rat was then anesthetized with intraperitoneal Nembutal (at 0.15 mL/100 g body weight). Magnetization for one second was performed to the rat chest under a magnetic flux density of 50 mT, followed by a 40-minute measurement of strength of the postmagnetization remanent magnetic field with a fluxmeter of flux-gate type. The apparatus was operated in such a way that the sample table passed over the probe once every 12 seconds. Magnetization and measurement of the remanent magnetic field of the lung was performed 1, 3, 14, and 28 days after RW exposure. By measuring the remanent magnetic field over 40 minutes postmagnetization, a curve indicating the decay constant can be obtained. Further, measurement of the remnant magnetic field strength for 2 min postmagnetization gave a nearly linear curve when plotted after logarithmic transformation. The point at which this curve intersected with the y-axis was designated B₀. When expressing the remnant magnetic field immediately after magnetization as B₀ and the decay constant as λ, the remnant magnetic field after t seconds of termination of external magnetization can be represented by the formula B = B₀e^-λt, and thus the decay constant (λ) was calculated based on this formula. In addition, the maximum strength of remanent magnetic field on each measurement day (t = 0 - minute value) was calculated with the value on Day 0 taken as 100%, on the basis of which clearance curves were prepared.

One, 3, 14 and 28 days after exposure, 6 rats were sacrificed a time (1D group, 3D group, 14D group, and 28D group, respectively). Rats were weighed once every week. During and after exposure, rats were intermittently monitored for any change in their appearance or condition.

Under Nembutal anesthesia, rats were sacrificed by exsanguination from the abdominal aorta and their lungs were resected. The resected lungs were ashed in a low-temperature asher (Plasma Asher, LTA-102, Yanaco Corp., Kyoto, Japan) over 24 hours.

The ashed specimen containing fibers was suspended in distilled water that had been filtered with a Minisart (Sartotius K. K., Tokyo, Japan) syringe filter unit in a weighing bottle. Fibers were collected on a Nuclepore filter (pore diameter, 0.2 μm) using a suction filter, and allowed to dry. At least 400 fibers were counted for each rat by use of a scanning electron microscope (BX41, Olympus Corp., Tokyo, Japan) at ×500 to ×2000 magnification. Fibers counted were those having an aspect ratio (ratio of length to width) of 3 or greater. The number of fibers in each of the three categories of length (L) (L ≤ 5, 5 < L ≤ 20, or L > 20) was obtained in accordance with the rules for fiber counting [17]. Among the fibers counted, World Health Organization (WHO) fibers – which have a length of longer than 5 μm and a width of shorter than 3 μm [2] – were also counted. The fiber number was then converted to the fiber number per weight of dried lung. The half-life of fibers in the rat lungs was calculated assuming that the geometric mean of the total fiber number/the total lung weight (fibers/mg) in the lungs of the 1D group was 100% [3].

Furthermore, the fiber size (length and width) was measured at ×500 to ×2000 magnification. In this measurement, fibers having a length of 0.47 μm or greater and a width of 0.05 μm or greater were included.

Three rats each were sacrificed 1, 3, 14, and 28 days after RW exposure. Their lungs were isolated and fixed in formalin, followed by observation of lung tissue by hematoxylin and eosin staining using a transmission electron microscope.

In the lung magnetometric evaluation, arithmetic mean values and standard deviations were calculated from data obtained for the RW-exposed and control groups of 6 rats each. Subsequently, Students' t-test was conducted.

In the biopersistence test, geometric mean and geometric standard deviation were calculated for the total fiber number, length and width. For length and width, at least 400 fibers in lungs per rat were counted in two experiments and the geometric mean value for 6 rats was calculated. One-way analysis of variance was performed and Scheffe's multiple comparison test was conducted.

In the lung magnetometric evaluation, the arithmetic mean (standard deviation, SD) of the total fiber concentration in the exposure chamber during the experiment was 650.2 (367.3) fibers/cm³, and 156.6 (104.7) fibers/cm³ for fibers with L > 20 μm. The arithmetic mean (SD) of the weight concentration was 170.4 (29.3) mg/m³.

In the biopersistence test, the arithmetic mean (SD) of the fiber concentration was 344.7 (161.6) fibers/cm³ for all fibers and 93.1 (50.2) fibers/cm³ for fibers with L > 20 μm. The arithmetic mean (SD) of the weight concentration was 100.0 (29.9) mg/m³.

In the pathological evaluation, the arithmetic mean (SD) of the fiber concentration was 390.7 (170.4) fibers/cm³ for all fibers and 95.0 (45.8) fibers/cm³ for fibers with L > 20 μm. The arithmetic mean (SD) of the weight concentration was 100.2 (26.4) mg/m³.

Assuming that the percentage of remanent magnetic field strength immediately after magnetization on each measurement day was 100%, the percentages of 40-minute postmagnetization period were calculated and plotted to construct relaxation curves. In both the RW-exposed and control groups, relaxation was rapid on all measurement days, as shown in Figure 2. No significant differences in decay were noted between the two groups on any of the study days. Between day 1 and day 14 there was an increase in the decay constant indicating an acceleration of relaxation during this time period. The decay constant during a 2-minute postmagnetization period did not significantly differ between the two groups on any measurement day (Figure 3).

The percentage of the remanent magnetic field strength immediately after magnetization (B₀) on each measurement day was calculated with the value obtained one day after exposure taken as 100%. The decay of B₀ shows the retention and clearance of the magnetic particles in the lung. Both the RW-exposed and control groups showed rapid magnetic particle clearance. In the RW-exposed group, however, magnetic particle clearance was impaired in tendency (Figure 4).

Table 1 shows the changes over time in the number of RW fibers that retained in lungs, and Figure 5 shows the percentage of the number of the retained fibers, calculated with the geometric mean of the 1D group taken as 100%. The total fiber number, fiber number by size, and WHO fiber number decreased over time in the observation period. The results of Scheffe's multiple comparisons showed that the fiber number in all categories significantly decreased in the 28D group as compared with the 1D group (p < 0.05) (Table 1).

The half-lives of RW fibers were calculated from an exponential approximation curve after the geometric mean of the 1D group was taken as 100%. The half-lives were 35 days for all fibers, 16 days for the fibers with L > 20 μm, and 35 days for WHO fibers. These findings indicate that the half-life of RW fibers with L > 20 μm was shorter (16 days) than that of all fibers or WHO fibers (35 days), showing that RW fibers have lower biopersistence.

As shown in Table 2, both length and width reduced over time in the observation period. Upon Scheffe's multiple comparisons, the 3D and 28D groups showed significantly shorter widths than the 1D group (p < 0.05) (Table 2).

An electron microscope image of the lung in the 28D group showed that macrophages retained morphologically almost normal nuclei and cytoplasm. Lung tissues did not show pulmonary fibrosis and were almost normal (data not shown).